GSM8045769: Sham_skull; Mus musculus; RNA-Seq
A total of 33 male C57BL/6J mice from sham, tMCAO and tMCAO+UVB groups were used for library preparation to perform scRNA-seq. After 1 day ischemia-reperfusion, mice were anesthetized, fixed and perfused with cooling PBS to remove the blood. The ischemic hemisphere (tMCAO ipsilateral brain) was immediately dissected. Meanwhile, the skullcap and meninges were harvested, respectively. The skullcap and meninges samples were minced using a scalpel for digestion with 2 mg/mL collagenase (Shanghai Life iLab Biotech Co., Ltd. China) for 1 h and 35 min, respectively, and red blood cells were lysed with 2 mL blood red cell lysing reagent (Biosharp, Labgic Technology Co., Ltd.). The brain tissue was digested with 10U/mL papain (Worthington Biochemical Corporation, USA) for 30 minutes. And, red blood cells were lysed with 4 mL blood red cell lysing reagent (Biosharp, Labgic Technology Co., Ltd.). Debris Removal Solution（130-109-398, Miltenyi Biotec）was used to remove fragments. After the single-cell suspension was obtained, automated cell counters (Countstar Rigel S2, Shanghai Ruiyu Biotechnology Co., LTD, China) was employed for cell number count (cell viability >80%). Single-cell suspensions from all tissues (10 μl each) were counted and the cell concentration was adjusted to achieve a cell recovery of approximately 10,000 cells.Single-cell suspensions were loaded onto the Chromium Controller using the Chromium Single Cell 3′ Library & Gel Bead Kit (10x Genomics, PN-1000268). The 3′ scRNA-seq libraries was established mainly following Chromium Single Cell 3' Reagent Kits User Guide (v3.1 Chemistry). Briefly, the gel bead emulsion was generated from the mixture of single cell suspension, gel beads and oils using the 10x Genomics chromium controller. Following droplet formation, the samples were transferred into PCR tubes. Reverse transcription was carried out using a T100 Thermal Cycler (Bio-Rad) according to the following protocol: incubation at 53℃ for 45 minutes, 85℃ for 5 minutes and finally held at 4℃. The resulting cDNA was then amplified and subjected to quality assessment using an Agilent Bioanalyzer 2100. The library was constructed by adding the P5 primer, Read 2 (primer site for sequencing read 2), Sample Index and P7 primer, respectively. Subsequently, the constructed library underwent quality control measures before being subjected to sequencing using the Illumina Novaseq 6000 platform (NovaSeq 6000 S4 Reagent Kit v1.5 with a 151-8-8-151 read setup). Details regarding sequencing depth and cell recovery are provided in Supplementary Table 2. Processing of scRNA-seq data was performed with the cellranger pipeline (v6.0.1, 10X Genomics) according to the manufacturer's instructions. Briefly, raw bcl files were de-multiplex using the cellranger mkfastq pipeline. Subequent read alignments and transcript counting were performed individually for each sample using cellranger count pipeline with default parameters.